Transfusion of Cd47^−/− RBCs into WT recipient mice results in retention of these cells in RP. (A) Representative flow cytometric plots (left panel) and the percentage (right panel) of CFSE-positive RBCs among total splenic RBCs of WT recipient mice at 2, 4, and 20 hours after IV injection of CFSE-labeled WT or Cd47^−/− RBCs. The data in the right panel are means ± SEM for 3 recipient mice. (B) Representative fluorescence microscopic images of frozen sections for the spleen of WT recipient mice at 2 hours after IV injection of CFSE-labeled WT or Cd47^−/− RBCs. Fluorescence images for CFSE (green) with or without DAPI (blue) are shown. Scale bar, 200 μm. (C) Representative fluorescence microscopic image of a frozen section for the spleen of a WT recipient mouse at 2 hours after IV injection of CFSE-labeled Cd47^−/− RBCs (green). The section was also subjected to immunohistofluorescence analysis with antibodies to F4/80 (red) and to laminin (white). Scale bar, 10 μm. Arrowheads and arrows indicate CFSE-labeled Cd47^−/− RBCs that are surrounded or not surrounded by F4/80-positive signals, respectively. (D) Representative TEM image of a spleen section from a WT recipient mouse at 2 hours after IV injection of Cd47^−/− RBCs. The white circles include RPMs that appear to be engaged in phagocytosis of RBCs (arrowheads). The arrows indicate RBCs that are localized independently of RPMs. Scale bar, 10 μm. (E) Representative TEM images of RP for WT recipient mice at 2 hours after IV injection of WT (upper left panel) or Cd47^−/− (lower left panel) RBCs. Asterisks indicate RBCs positioned independently of RPMs. Scale bar, 5 μm. The percentage area occupied by RBCs in such TEM images (23 × 23 μm²) of RP in WT recipient mice is also revealed (right panel). The data are means ± SEM for 30 TEM images from 3 mice. ∗∗P < .01 by Student t test (panels A,E). DAPI, 4′,6-diamidino-2-phenylindole; FSC-H, forward scatter height; NS, not significant; RP, red pulp; WP, white pulp.